PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the world?s deadliest disease, affects an estimated one-third of the world?s population. There is an urgent need to deepen our understanding of Mtb physiology so that new drug targets can be identified and exploited, thereby resulting in improved human health. Mtb has evolved to utilize the host?s cholesterol as a main carbon source when enveloped by the macrophage, increasing its persistence and pathogenicity. The genes mftC, mftD, and mftE were shown to be critical for Mtb growth on cholesterol and belong to the mycofactocin biosynthetic pathway, which is encoded by the gene cluster mftABCDE. Mycofactocin has been proposed to be a novel, peptide-derived, redox cofactor, however the final structure and physiological role of the molecule has yet to be determined. It is known that the first step of mycofactocin biosynthesis begins with the MftC catalyzed decarboxylation of the conserved C-terminal tyrosine on MftA, assisted by protein interactions with the peptide chaperone MftB. Characterization of mycofactocin biosynthesis is a straightforward approach towards evaluating a potentially druggable pathway while advancing our understanding of a critical component in Mtb physiology and constitutes the long-term goal. The overall objective of this application is to apply biochemical and biophysical concepts and techniques to study mycofactocin biosynthesis by elucidating the chemical mechanism of MftC catalysis, resolving the sequences required for protein interactions within the pathway, and to elucidate the chemistry and the product of MftD catalysis. The central hypothesis is that mycofactocin is synthesized from extensive modification of the C-terminal tyrosine on MftA by MftC and MftD. These modifications require fine tuning of the electrochemical environments of the redox centers in MftC, a flavin dependent reaction catalyzed by MftD, and specific protein-protein and protein-peptide interactions. Considering that MftC, MftD, and MftE are required for Mtb, leaving the mycofactocin biosynthetic pathway uncharacterized prevents potentially exploiting them as therapeutic targets for Mtb. Guided by strong preliminary data, the hypothesis will be tested by the three specific aims: 1) Determine the mechanism of MftC catalyzed oxidative decarboxylation of MftA, 2) Resolve sequence motifs involved in interactions with the mycofactocin biosynthetic pathway peptide and peptide chaperone, and 3) Discover the function of MftD in mycofactocin biosynthesis. The research is innovative, because it departs from the status quo by elucidating the biosynthesis of a potentially new, peptide derived, redox cofactor and by providing sequence and spatial resolution of the little understood protein interactions that are required for mycofactocin biosynthesis. Advances made through the characterization of the mycofactocin biosynthetic pathway will provide valuable information about essential Mtb proteins and will increase our understanding of Mtb physiology. Such knowledge has the potential to be leveraged for therapeutic development for the treatment of world?s deadliest disease.